Innate immune responses against glioma (GBM) are poorly understood. Most studies have focused on adaptive T cell immune responses. Innate immune responses are thought to be needed primarily, to activate T cell responses, rather than mediate direct cytotoxicity against tumors. Recently we showed that NK cells inhibit GBM progression, and exert powerful anti- GBM cytotoxicity. In turn, to evade NK- killing GBMs produce potent inhibitors of NK cells. Having established that NK cells inhibit GBM growth and invasion, we will evaluate the complex network of innate immune cells and signaling pathways responsible for this powerful anti-GBM response. Our data support the hypothesis that other innate immune cells, besides NK cells, are necessary for the powerful NK-mediated anti-GBM responses, as GR1 depletion abolishes NK- mediated GBM killing. In AIM 1 will identify the network of innate immune cells required to inhibit GBM progression. Our preliminary data show that Myd88 signaling is necessary for trafficking of innate immune cells to the tumor microenvironment and control tumor growth. In AIM 2 we will test the hypothesis that Myd88 transduces cellular responses to TLR9, IL18, and/or IL33 signaling in cells of the myeloid lineage within the tumor microenvironment. We will assess in which cells Myd88 signaling is needed for NK cells to kill GBM cells. Preliminary data suggest that the cGAS-STING-IFN? pathway is also necessary for NK-mediated GBM killing. In AIM 3 we will test the hypothesis that signaling via the cGAS-STING-IRF3-IFN? pathway on pDCs -or other myeloid cells- is necessary for full cytotoxic NK activation. We propose to test whether both pathways (Myd88 and STING) are necessary for innate immune-mediated inhibition of GBM progression. In summary, our proposal will ascertain the network of innate immune cells and signaling pathways that jointly inhibit GBM progression. In addition, the work proposed will also establish if the two innate signaling pathways (Myd88 and STING) converge to stimulate malignant GBM killing. The complex innate immune network and its signaling through Myd88 and STING to inhibit brain tumor progression solely via innate immunity have not yet been elucidated. Finally, we will test therapeutic combinations of a conditional cytotoxic-immune stimulatory approach (Ad-TK Ad-Flt3L) with the activation of innate immune signaling pathways (Myd88 and STING) in genetically engineered mouse models of GBM. In the long term, we aim to develop novel translational clinical trials, as we achieved earlier for gene/immune-therapeutic treatment of human gliomas using Ad-TK and Ad-Flt3L (NCT01811992).